Diffusion enhancing compounds and their use alone or with thrombolytics

ABSTRACT

The subject invention relates to diffusion enhancing compounds and their use alone or with thrombolytic agents for the treatment of disorders resulting from the formation of a thrombus such as a myocardial infarction or stroke.

This application is a continuation of U.S. patent application Ser. No.16/193,762 filed on Nov. 16, 2018, which is a continuation of U.S.patent application Ser. No. 12/801,726 filed on Jun. 22, 2010, whichclaims benefit of, and priority from, U.S. Provisional Application Ser.No. 61/213,575 filed on Jun. 22, 2009, the entire contents of which arehereby incorporated by reference in this application.

FIELD OF THE INVENTION

The subject invention relates to diffusion enhancing compounds and theiruse, either alone or with thrombolytic agents, for the treatment ofdisorders resulting from the formation of a thrombus such as amyocardial infarction or stroke.

BACKGROUND OF THE INVENTION

A thrombus is the inappropriate activation of the hemostatic process inan uninjured or slightly injured vessel. A thrombus in a large bloodvessel (mural thrombus) will decrease blood flow through that vessel. Ina small blood vessel (occlusive thrombus), blood flow may be completelycut-off resulting in death of tissue supplied by that vessel. If athrombus dislodges and becomes free-floating, it is termed as anembolus.

Some of the conditions which elevate risk of blood clots developinginclude atrial fibrillation (a form of cardiac arrhythmia), heart valvereplacement, a recent heart attack, extended periods of inactivity (seedeep venous thrombosis below), and genetic or disease-relateddeficiencies in the blood's clotting abilities.

Blood clot prevention and treatment reduces the risk of stroke, heartattack and pulmonary embolism. Heparin and warfarin are often used toinhibit the formation and growth of existing thrombi; they are able todecrease blood coagulation by inhibiting vitamin K epoxide reductase, anenzyme needed to form mature clotting factors.

Acute ischemic stroke (AIS) is a potentially devastating disease thatgoes untreated in greater than 95% of patients. Acute ischemic stroke isestimated to affect more than 700,000 patients each year in the USA and15 million worldwide [1,2]. New pharmacological therapeutics that canreduce the clinical deficits associated with AIS are needed. Ischemicstroke results from an obstruction within a blood vessel supplying bloodto the brain.

Hemorrhagic stroke accounts for about 17 percent of stroke cases. Itoccurs when a weakened blood vessel ruptures.

Tissue plasminogen activator (tPA) is a protein thrombolytic agent(clot-busting drug). It is approved for use in certain patients having aheart attack or stroke. The drug can dissolve blood clots, which causemost heart attacks and strokes. tPA is the only drug approved by theU.S. Food and Drug Administration for the acute (urgent) treatment ofischemic stroke. Specifically, it is approved for the treatment ofischemic stroke in the first three hours after the start of symptoms[3].

If given promptly, tPA can significantly reduce the effects of ischemicstroke and reduce permanent disability. However, a time delay instarting tPA treatment often occurs because, when a patient presentswith stroke-like symptoms, it is not immediately apparent whether thestroke has been caused by blood clots (ischemic stroke) or by a rupturedblood vessel (hemorrhagic stroke). tPA can only be given for ischemicstrokes; therefore, the type of stroke must be determined before tPA isadministered.

Although over 80% of all strokes are ischemic strokes, tPA or anythrombolytic, cannot be given immediately since it is possible that itcould cause the hemorrhagic strokes to produce even worse effects.Determining whether a given patient has suffered a hemorrhagic orischemic stroke is a time-consuming diagnosis which stands as a “gate”to immediate treatment. That, coupled with the fact that tPA must begiven within 3 hours of the first symptoms, has resulted in only a smallfraction of stroke patients receiving tPA.

tPA is effective in numerous preclinical models of acute ischemic strokeincluding the rabbit small clot embolic stroke model (RSCEM), [4] auseful tool and possibly a predictor of effective treatments that mayeventually translate into functional efficacy in human clinical trials[2,4-7]. The primary endpoint used when assessing treatment efficacy inthe RSCEM is functional behavior, which is based upon motor functioncomponents of the National Institute of Health Stroke Scale (NIHSS) forstroke in humans [8, 9].

Cerebral edema is the presence of excess fluid within either the cellsor the extracellular spaces of the brain. This disorder also causesbrain swelling and a rise in intracranial pressure. Head injuries,encephalitis, abscesses, lack of oxygen, tumors, strokes, and toxicagents are the most common causes of cerebral edema. Current treatmentapproaches to cerebral edema can include mannitol, diuretics andcorticosteroids. One of the main corticosteroids used is dexamethasone(Decadron).

Carotenoids are a class of hydrocarbons consisting of isoprenoid units.The backbone of the molecule consists of conjugated carbon-carbon doubleand single bonds, and can have pendant groups. Carotenoids such ascrocetin and trans sodium crocetinate (TSC) are known to increase thediffusivity of oxygen in water.

U.S. Pat. No. 6,060,511 relates to trans sodium crocetinate (TSC) andits uses. The patent covers various uses of TSC such as improving oxygendiffusivity and treatment of hemorrhagic shock.

U.S. patent application Ser. No. 10/647,132 relates to synthesis methodsfor making bipolar trans carotenoids (BTC), including bipolar transcarotenoid salts (BTCS), and methods of using them.

U.S. patent application Ser. No. 11/361,054 relates to improved BTCsynthesis methods and novel uses of the BTC.

U.S. patent application Ser. No. 12/081,236 relates to the use ofbipolar trans carotenoids as a pretreatment and in the treatment ofperipheral vascular disease.

U.S. application Ser. No. 12/289,713 relates to a new class oftherapeutics that enhance small molecule diffusion.

SUMMARY OF THE INVENTION

The subject invention relates to a method of treating a mammal having anischemic stroke, myocardial infarction, pulmonary embolism, or deep veinthrombosis comprising; administering a diffusion enhancing compound tosaid mammal, and administering a thrombolytic agent to said mammal. Theinvention also relates to a method of treating a mammal having a strokewhere it is unknown whether the stroke is an ischemic stroke or ahemorrhagic stroke comprising: i) administering a diffusion enhancingcompound to said mammal, ii) determining whether the stroke is anischemic stroke, and if so determined, iii) administering a thrombolyticagent to said mammal.

The invention also relates to a method of treating a mammal having ahemorrhagic stroke, cerebral edema, or TIA comprising administering adiffusion enhancing compound to said mammal.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides the quantal curve for the effect of TSC on behavior.

FIG. 2 provides a graphical representation of the effects of tPA onbehavior and the Group P₅₀ value when given three hours followingembolization.

FIG. 3 is a bar graph for single drug treatment.

FIG. 4 is a bar graph for combination therapy versus single drugtherapy.

FIG. 5 is a bar graph concerning various treatments at one hour.

FIG. 6 depicts examples of hematomas from Collagenase-Injection ICHModel.

FIG. 7 is a bar graph of the effect of TSC on hematoma size.

FIG. 8 is a bar graph of the effect of TSC on hemorrhagic volume.

FIG. 9 is a bar graph of the effect of TSC on tissue edema.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention relates to diffusion enhancing compounds and theiruse with thrombolytic agents for the treatment of disorders resultingfrom the formation of a thrombus such as a myocardial infarction orstroke.

Compounds and Compositions of the Invention Thrombolytics

Thrombolysis is used in myocardial infarction (heart attack), ischemicstrokes, deep vein thrombosis and pulmonary embolism to clear a blockedartery, i.e. a thrombus, and avoid permanent damage to the affectedtissue (e.g. myocardium, brain, leg) and death. A less frequent use isto clear blocked catheters that are used in long-term medical therapy.

It should be noted that thrombolytic therapy in hemorrhagic strokes iscontraindicated, as its use in that situation would prolong bleedinginto the intracranial space and cause further damage.

The thrombolytic drugs include:

-   -   tissue plasminogen activator—t-PA—alteplase (Activase)    -   reteplase (Retavase)    -   tenecteplase (TNKase)    -   anistreplase (Eminase)    -   streptokinase (Kabikinase, Streptase)    -   urokinase (Abbokinase)

These drugs are most effective if administered immediately after it hasbeen determined they are clinically appropriate. The drugs can be givenin combination with intravenous heparin, or low molecular weightheparin, which are anticoagulant drugs.

Diffusion Enhancing Compounds

The diffusion enhancing compounds of the invention include thosecompounds described in U.S. Ser. No. 10/647,132, U.S. Ser. No.11/361,054, U.S. Ser. No. 12/081,236 and U.S. Ser. No. 12/289,713, eachof which is hereby incorporated by reference in its entirety.

Included are bipolar trans carotenoid compounds having the formula:

YZ-TCRO—ZY

where:

Y=a cation

Z=a polar group which is associated with the cation, and

TCRO=trans carotenoid skeleton,

such as TSC.

More specifically, the subject invention relates to trans carotenoidsincluding trans carotenoid diesters, dialcohols, diketones and diacids,bipolar trans carotenoids (BTC), and bipolar trans carotenoid salts(BTCS) compounds and synthesis of such compounds having the structure:

YZ-TCRO—ZY

where:

-   -   Y (which can be the same or different at the two ends)=H or a        cation other than H, preferably Na⁺ or K⁺ or Li⁺. Y is        advantageously a monovalent metal ion. Y can also be an organic        cation, e.g., R₄N⁺, R₃S⁺, where R is H, or C_(n)H_(2n+1) where n        is 1-10, advantageously 1-6. For example, R can be methyl,        ethyl, propyl or butyl.    -   Z (which can be the same or different at the two ends)=polar        group which is associated with H or the cation. Optionally        including the terminal carbon on the carotenoid (or carotenoid        related compound), this group can be a carboxyl (COO⁻) group or        a CO group (e.g. ester, aldehyde or ketone group), or a hydroxyl        group. This group can also be a sulfate group (OSO₃ ⁻) or a        monophosphate group (OPO₃ ⁻), (OP(OH)O₂ ⁻), a diphosphate group,        triphosphate or combinations thereof. This group can also be an        ester group of COOR where the R is C_(n)H_(2n+1).    -   TCRO=trans carotenoid or carotenoid related skeleton        (advantageously less than 100 carbons) which is linear, has        pendant groups (defined below), and typically comprises        “conjugated” or alternating carbon-carbon double and single        bonds (in one embodiment, the TCRO is not fully conjugated as in        a lycopene). The pendant groups (X) are typically methyl groups        but can be other groups as discussed below. In an advantageous        embodiment, the units of the skeleton are joined in such a        manner that their arrangement is reversed at the center of the        molecule. The 4 single bonds that surround a carbon-carbon        double bond all lie in the same plane. If the pendant groups are        on the same side of the carbon-carbon double bond, the groups        are designated as cis (also known as “Z”); if they are on the        opposite side of the carbon-carbon bond, they are designated as        trans (also known as “E”). Throughout this case, the isomers        will be referred to as cis and trans.    -   The compounds of the subject invention are trans. The cis isomer        typically is a detriment—and results in the diffusivity not        being increased. In one embodiment, a cis isomer can be utilized        where the skeleton remains linear. The placement of the pendant        groups can be symmetric relative to the central point of the        molecule or can be asymmetric so that the left side of the        molecule does not look the same as the right side of the        molecule either in terms of the type of pendant group or their        spatial relationship with respect to the center carbon.

The pendant groups X (which can be the same or different) are hydrogen(H) atoms, or a linear or branched hydrocarbon group having 10 or lesscarbons, advantageously 4 or less, (optionally containing a halogen), ora halogen. X could also be an ester group (COO—) or an ethoxy/methoxygroup. Examples of X are a methyl group (CH₃), an ethyl group (C₂H₅), aphenyl or single aromatic ring structure with or without pendant groupsfrom the ring, a halogen-containing alkyl group (C1-C10) such as CH₂Cl,or a halogen such as Cl or Br or a methoxy (OCH₃) or ethoxy (OCH₂CH₃).The pendant groups can be the same or different but the pendant groupsutilized must maintain the skeleton as linear.

Although many carotenoids exist in nature, carotenoid salts do not.Commonly-owned U.S. Pat. No. 6,060,511 hereby incorporated by referencein its entirety, relates to trans sodium crocetinate (TSC). The TSC wasmade by reacting naturally occurring saffron with sodium hydroxidefollowed by extractions that selected primarily for the trans isomer.

The presence of the cis and trans isomers of a carotenoid or carotenoidsalt can be determined by looking at the ultraviolet-visible spectrumfor the carotenoid sample dissolved in an aqueous solution. Given thespectrum, the value of the absorbence of the highest peak which occursin the visible wave length range of 380 to 470 nm (the number dependingon the solvent used and the chain length of the BTC or BTCS. Theaddition of pendant groups or differing chain lengths will change thispeak absorbance but someone skilled in the art will recognize theexistence of an absorbance peak in the visible range corresponding tothe conjugated backbone structure of these molecules.) is divided by theabsorbency of the peak which occurs in the UV wave length range of 220to 300 nm can be used to determine the purity level of the trans isomer.When the trans carotenoid diester (TCD) or BTCS is dissolved in water,the highest visible wave length range peak will be at between 380 nm to470 nm (depending on the exact chemical structure, backbone length andpendant groups) and the UV wave length range peak will be between 220 to300 nm. According to M. Craw and C. Lambert, Photochemistry andPhotobiology, Vol. 38 (2), 241-243 (1983) hereby incorporated byreference in its entirety, the result of the calculation (in that casecrocetin was analyzed) was 3.1, which increased to 6.6 afterpurification.

Performing the Craw and Lambert analysis, using a cuvette designed forUV and visible wavelength ranges, on the trans sodium salt of crocetinof commonly owned U.S. Pat. No. 6,060,511 (TSC made by reactingnaturally occurring saffron with sodium hydroxide followed byextractions which selected primarily for the trans isomer), the valueobtained averages about 6.8. Performing that test on the synthetic TSCof the subject invention, that ratio is greater than 7.0 (e.g. 7.0 to8.5), advantageously greater than 7.5 (e.g. 7.5-8.5), mostadvantageously greater than 8. The synthesized material is a “purer” orhighly purified trans isomer.

Formulation and Administration of the Compounds and Compositions of theInvention

A detailed description of formulation and administration of diffusingenhancing compounds can be found in commonly owned applications U.S.Ser. No. 12/081,236 and U.S. Ser. No. 12/289,713, each of which ishereby incorporated by reference in its entirety.

A diffusion enhancing compound such as TSC can be administered byvarious routes. For example, the compound which can be formulated withother compounds including excipients, can be administered at the properdosage as an intravenous injection or infusion, an intramuscularinjection, or in an oral form.

The IV injection route is an advantageous route for giving TSC for theuses of the subject application since the patient may well beunconscious. Typically, a diffusion enhancing compound such as TSC isadministered as soon as possible if a thrombus is believed present or ifthe patient is hemorrhaging.

Cyclodextrins

In order to administer some pharmaceuticals, it is necessary to addanother compound which will aid in increasing theabsorption/solubility/concentration of the active pharmaceuticalingredient (API). Such compounds are called excipients, andcyclodextrins are examples of excipients. Cyclodextrins are cycliccarbohydrate chains derived from starch. They differ from one another bythe number of glucopyranose units in their structure. The parentcyclodextrins contain six, seven and eight glucopyranose units, and arereferred to as alpha, beta and gamma cyclodextrins respectively.Cyclodextrins were first discovered in 1891, and have been used as partof pharmaceutical preparations for several years.

Cyclodextrins are cyclic (alpha-1,4)-linked oligosaccharides ofalpha-D-glucopyranose containing a relatively hydrophobic central cavityand hydrophilic outer surface. In the pharmaceutical industry,cyclodextrins have mainly been used as complexing agents to increase theaqueous solubility of poorly water-soluble drugs, and to increase theirbioavailability and stability. In addition, cyclodextrins are used toreduce or prevent gastrointestinal or ocular irritation, reduce oreliminate unpleasant smells or tastes, prevent drug-drug ordrug-additive interactions, or even to convert oils and liquid drugsinto microcrystalline or amorphous powders.

Although the BTC compounds are soluble in water, the use of thecyclodextrins can increase that solubility even more so that a smallervolume of drug solution can be administered for a given dosage.

There are a number of cyclodextrins that can be used with the Compoundsof the Invention. See for example, U.S. Pat. No. 4,727,064, herebyincorporated by reference in its entirety. Advantageous cyclodextrinsare γ-cyclodextrin, 2-hydroxylpropyl-γ-cyclodextrin and2-hydroxylpropyl-β-cyclodextrin, or other cyclodextrins which enhancethe solubility of the BTC.

The use of gamma-cyclodextrin with TSC increases the solubility of TSCin water by 3-7 times. Although this is not as large a factor as seen insome other cases for increasing the solubility of an active agent with acyclodextrin, it is important in allowing for the parenteraladministration of TSC in smaller volume dosages to humans (or animals).Dosages of TSC and gamma-cyclodextrin have resulted in aqueous solutionscontaining as much as 44 milligrams of TSC per ml of solution, with anadvantageous range of 20-30 mg/ml of solution. The solutions need not beequal-molar. The incorporation of the gamma cyclodextrin also allows forTSC to be absorbed into the blood stream when injected intramuscularly.Absorption is quick, and efficacious blood levels of TSC are reachedquickly (as shown in rats).

The cyclodextrin formulation can be used with other trans carotenoidsand carotenoid salts. The subject invention also includes novelcompositions of carotenoids which are not salts (e.g. acid forms such ascrocetin, crocin or the intermediate compounds noted above) and acyclodextrin. In other words, trans carotenoids which are not salts canbe formulated with a cyclodextrin. Mannitol can be added for osmolality,or the cyclodextrin BTC mixture can be added to isotonic saline (seebelow).

The amount of the cyclodextrin used is that amount which will containthe trans carotenoid but not so much that it will not release the transcarotenoid. Advantageously, the ratio of cyclodextrin to BTC, e.g., TSC,is 4 to 1 or 5 to 1. See also U.S. Patent Application No. 61/350,804,the content of which is hereby incorporated by reference in itsentirety.

Cyclodextrin-Mannitol

A trans carotenoid such as TSC can be formulated with a cyclodextrin asnoted above and a non-metabolized sugar such as mannitol (e.g.d-mannitol to adjust the osmotic pressure to be the same as that ofblood). Solutions containing over 20 mg TSC/ml of solution can be madethis way. This solution can be added to isotonic saline or to otherisotonic solutions in order to dilute it and still maintain the properosmolality.

Mannitol/Acetic Acid

A BTCS such as TSC can be formulated with mannitol such as d-mannitol,and a mild buffering agent such as acetic acid or citric acid to adjustthe pH. The pH of the solution should be around 8 to 8.5. It should beclose to being an isotonic solution, and, as such, can be injecteddirectly into the blood stream.

Water+Saline

A BTCS such as TSC can be dissolved in water (advantageously injectablewater). This solution can then be diluted with water, normal saline,Ringer's lactate or phosphate buffer, and the resulting mixture eitherinfused or injected.

Buffers

A buffer such as glycine, bicarbonate, or sodium carbonate can be addedto the formulation at a level of about 50 mM for stability of the BCTsuch as TSC.

TSC and Gamma-Cyclodextrin

The ratio of TSC to cyclodextrin is based on TSC:cyclodextrin solubilitydata. For example, 20 mg/ml TSC, 8% gamma cyclodextrin, 50 mM glycine,2.33% mannitol with pH 8.2+/−0.5, or 10 mg/ml TSC and 4% cyclodextrin,or 5 mg/ml and 2% cyclodextrin. The ratios of these ingredients can bealtered somewhat, as is obvious to one skilled in this art.

Mannitol can be used to adjust osmolality and its concentration variesdepending on the concentration of other ingredients. The glycine is heldconstant. TSC is more stable at higher pHs. pH of around 8.2+/−0.5 isrequired for stability and physiological compatibility. The use ofglycine is compatible with lyophilization. Alternatively, the TSC andcyclodextrin is formulated using a 50 mM bicarbonate buffer in place ofthe glycine.

Endotoxin Removal of Gamma-Cyclodextrin

Commercially available pharmaceutical grade cyclodextrin has endotoxinlevels that are incompatible with intravenous injection. The endotoxinlevels must be reduced in order to use the cyclodextrin in a BTCformulation intended for intravenous injection.

After it is determined that a thrombus is present, a therapeuticallyeffective amount, i.e. a clot dissolving amount, of the thrombolyticagent such as tPA, can also be administered. Formulation ofthrombolytics is well known to those skilled in the art. A thrombolyticsuch as tPA, is typically administered via IV injection. If a diffusionenhancing drug has been administered, the advantage of administration ofa thrombolytic is highest within the first ninety minutes, but canextend up to 6, 9 or even 12 hours after the start of symptoms.

Thrombolytic and/or diffusion enhancing drugs also can be given incombination with intravenous heparin, or low molecular weight heparin,which are anticoagulant drugs. Heparin and warfarin are often used toinhibit the formation and growth of existing thrombi.

In one embodiment, the thrombolytic agent is formulated together withthe diffusion enhancing compound for IV administration.

Uses of the Compounds and Compositions of the Invention

A diffusion enhancing compound such as trans sodium crocetinate (TSC)can be administered either alone or in combination with the thrombolyticsuch as tissue plasminogen activator (tPA), to reduce deficitsassociated with a thrombosis.

Stroke

For a given isolated blood vessel, blood flow to the brain tissue can behampered in two ways:

-   -   1. the vessel clogs within (ischemic stroke)    -   2. the vessel ruptures, causing blood to leak into the brain        (hemorrhagic stroke)

A beneficial treatment for stroke would be:

-   -   (1) A drug which can be used for treating either a hemorrhagic        stroke or an ischemic stroke, or    -   (2) A drug which can increase the window for giving a        thrombolytic, e.g. the approved 3-hour window of time for giving        tPA.

Ischemic Stroke

Ischemic stroke accounts for about 83 percent of all cases. Ischemicstrokes occur as a result of an obstruction within a blood vesselsupplying blood to the brain. The underlying condition for this type ofobstruction is the development of fatty deposits lining the vesselwalls. This condition is called atherosclerosis. These fatty depositsare associated with two types of obstruction:

Cerebral thrombosis refers to a thrombus (blood clot) that develops atthe clogged part of the vessel.

Cerebral embolism refers generally to a blood clot that forms at anotherlocation in the circulatory system, usually the heart and large arteriesof the upper chest and neck. A portion of the blood clot breaks loose,enters the bloodstream and travels through the brain's blood vesselsuntil it reaches vessels too small to let it pass. A second importantcause of embolism is an irregular heartbeat, known as atrialfibrillation. It creates conditions where clots can form in the heart,dislodge and travel to the brain.

Also called TIAs, transient ischemic attacks are minor or warningstrokes. In a TIA, conditions indicative of an ischemic stroke arepresent and the typical stroke warning signs develop. However, thesymptoms occur for a short time and tend to resolve through normalmechanisms. Even though the symptoms disappear after a short time, TIAsmay be indicators of a possible major stroke. Steps should be takenimmediately to prevent an ischemic stroke. A patient showing signs of aTIA or at risk of a stroke should be given a diffusion enhancingcompound such as TSC, e.g., by IV injection or orally at a dosage in therange of 0.1-2 mg/kg.

In order to find new drugs for stoke victims, different animal models ofischemic stroke are used. In one model, blood vessels (middle cerebralartery, two carotid arteries) are ligated for a period of 2 hours. Theligature is then removed, a drug is given, and the animals (rats in thiscase) are sacrificed after 24 hours. The brain sections are stained andexamined in order to determine the amount of damaged (ischemic) tissue.With this model, it was found that a TSC dosage of 0.1 mg/kg in the ratsproduced a profound (about 60%) reduction in the amount of ischemictissue.

In a rat model of hemorrhagic shock, in which the enzyme collagenase isused to cause the blood vessels to leak into the brain, it was foundthat the administration of 0.1 mg/kg of TSC did not increase the amountof blood that hemorrhages into the brain. In fact, it caused a decreasein that hemorrhage volume. Of perhaps more importance, it was found thatTSC reduced the amount of edema caused by the hemorrhagic stroke byabout 50%. Thus, TSC appears to be a drug which meets category (1)above: a drug which can be used on either type of stroke without fear ofcausing further damage.

The combination of TSC and tPA effectively improves functional behaviorusing the RSCEM model discussed below. RSCEM is produced by injection ofblood clots into the cerebral vasculature of a rabbit to producecerebral ischemia resulting in behavioral deficits that can be measuredquantitatively using a dichotomous rating scale and a statisticalquantal analysis technique.

Example 1 below shows that TSC administration significantly improvesclinical rating scores when administered within 1 hour of embolizationin the RSCEM model. Moreover, the study shows that TSC can beadministered safely in combination with the thrombolytic tPA and thatcombination therapy also produces a significant functional behavioralimprovement in embolized rabbits. Simultaneous administration of TSC andtPA is beneficial for treating heart attacks caused by clots, as isgiving TSC alone.

The early use of a diffusion enhancing compound such as TSC can increasethe window of opportunity of giving a thrombolytic agent such as tPAlater in order to treat ischemic strokes. The data teaches that TSC canextend the treatment window for tPA to at least 3 hours in the RSCEMmodel, a time at which tPA alone is ineffective in this animal model.This time is believed to be multiplied by a factor of 3 to 4 in humans.Thus, if a diffusion enhancing compound such as TSC is given to a humanwithin the first 3-4 hours after the first stoke symptoms, then athrombolytic agent such as tPA can be given 9 or even up to 12 hoursafter the first stroke symptoms. A patient showing signs of an ischemicstroke should be given a diffusion enhancing compound such as TSC, e.g.,by IV injection or infusion, or orally, at a dosage in the range of0.1-2 mg/kg. Treatment with TSC alone is also an effective treatment forstroke.

Hemorrhagic Stroke

Hemorrhagic stroke accounts for about 17 percent of stroke cases. Itresults from a weakened vessel that ruptures and bleeds into thesurrounding brain. The blood accumulates and compresses the surroundingbrain tissue. The two types of hemorrhagic strokes are intracerebralhemorrhage or subarachnoid hemorrhage.

Hemorrhagic stroke occurs when a weakened blood vessel ruptures. Twotypes of weakened blood vessels usually cause hemorrhagic stroke:aneurysms and arteriovenous malformations (AVMs). An aneurysm is aballooning of a weakened region of a blood vessel. If left untreated,the aneurysm continues to weaken until it ruptures and bleeds into thebrain. An arteriovenous malformation (AVM) is a cluster of abnormallyformed blood vessels. Any one of these vessels can rupture, also causingbleeding into the brain.

A diffusion enhancing compound such as a BTCS compounds (e.g. TSC), canbe used in treatment of hemorrhagic stroke. The compound can beadministered by various routes, including IV injection or infusion ororally. The IV injection or infusion route is an advantageous route forgiving a diffusion enhancing compound for hemorrhagic stroke since thepatient may well be unconscious. Typically, a diffusion enhancingcompound such as TSC is administered as soon as possible if the patientis hemorrhaging, but can also be given after the hemorrhage hassubsided. A patient showing signs of a hemorrhagic stroke should begiven a diffusion enhancing compound such as TSC, e.g., by IV injectionor infusion, or orally, at a dosage in the range of 0.1-2 mg/kg.

Cerebral Edema

Cerebral edema is an excess accumulation of water in the intracellularand/or extracellular spaces of the brain. Four types of cerebral edemahave been distinguished:

(1) Vasogenic Cerebral Edema

This is due to a breakdown of tight endothelial junctions which make upthe blood-brain barrier (BBB). This allows normally excludedintravascular proteins and fluid to penetrate into cerebral parenchymalextracellular space. Once plasma constituents cross the BBB, the edemaspreads; this may be quite fast and widespread. As water enters whitematter it moves extracellularly along fiber tracts and can also affectthe gray matter. This type of edema is seen in response to trauma,tumors, focal inflammation, late stages of cerebral ischemia andhypertensive encephalopathy.Some of the mechanisms contributing to BBB dysfunction are: physicaldisruption by arterial hypertension or trauma, tumor-facilitated releaseof vasoactive and endothelial destructive compounds (e.g. arachidonicacid, excitatory neurotransmitters, eicosanoids, bradykinin, histamineand free radicals). Some of the special subcategories of vasogenic edemainclude:

-   -   A. Hydrostatic Cerebral Edema    -   This form of cerebral edema is seen in acute, malignant        hypertension. It is thought to result from direct transmission        of pressure to cerebral capillary with transudation of fluid        into the extra-cellular fluid from the capillaries.    -   B. Cerebral Edema from Brain Cancer    -   Cancerous glial cells (glioma) of the brain can increase        secretion of vascular endothelial growth factor (VEGF) which        weakens the junctions of the blood-brain barrier. Dexamethasone        (a corticosteroid compound) can be of benefit in reducing VEGF        secretion.    -   C. High Altitude Cerebral Edema    -   High altitude cerebral edema (or HACE) is a severe form of        (sometimes fatal) altitude sickness. HACE is the result of        swelling of brain tissue from leakage of fluids from the        capillaries due to the effects of hypoxia on the        mitochondria-rich endothelial cells of the blood-brain barrier.    -   Symptoms can include headache, loss of coordination (ataxia),        weakness, and decreasing levels of consciousness including        disorientation, loss of memory, hallucinations, psychotic        behavior, and coma. It generally occurs after a week or more at        high altitude. Severe instances can lead to death if not treated        quickly. Immediate descent is a necessary life-saving measure        (2,000-4,000 feet). There are some medications (e.g.        dexamethasone) that may be prescribed for treatment but these        require proper medical training in their use. Anyone suffering        from HACE must be evacuated to a medical facility for proper        follow-up treatment.    -   Climbers may also suffer high altitude pulmonary edema (HAPE),        which affects the lungs. While not as life threatening as HACE        in the initial stages, failure to descend to lower altitudes or        receive medical treatment can also lead to death.

(2) Cytotoxic Cerebral Edema

In this type of edema the BBB remains intact. This edema is due to thederangement in cellular metabolism resulting in inadequate functioningof the sodium and potassium pump in the glial cell membrane. As a resultthere is cellular retention of sodium and water. There are swollenastrocytes in gray and white matter. Cytoxotic edema is seen withvarious intoxications (dinitrophenol, triethyltin, hexachlorophene,isoniazid), in Reye's syndrome, severe hypothermia, early ischemia,encephalopathy, early stroke or hypoxia, cardiac arrest, pseudotumorcerebri, and cerebral toxins.

(3) Osmotic Cerebral Edema

Normally cerebral-spinal fluid (CSF) and extracellular fluid (ECF)osmolality of the brain is slightly greater than that of plasma. Whenplasma is diluted by excessive water intake (or hyponatremia), syndromeof inappropriate antidiuretic hormone secretion (SIADH), hemodialysis,or rapid reduction of blood glucose in hyperosmolar hyperglycemic state(HHS), formerly hyperosmolar non-ketotic acidosis (HONK), the brainosmolality will then exceed the serum osmolality creating an abnormalpressure gradient down which water will flow into the brain causingedema.

(4) Interstitial Cerebral Edema

Interstitial cerebral edema occurs in obstructive hydrocephalus. Thisform of edema is due to rupture of CSF-brain barrier: permits CSF topenetrate brain and spread in the extracellular space of white matter.Differentiated from vasogenic edema in that fluid contains almost noprotein.

A diffusion enhancing compound such as a BTCS compounds (e.g. TSC), canbe used in treatment of cerebral edema. The compound can be administeredby various routes, including IV injection or orally. The IV injectionroute is an advantageous route for giving TSC for cerebral edema sincethe patient may well be unconscious. Typically, a diffusion enhancingcompound such as TSC is administered as soon as a cerebral edema isdetected. A patient showing signs of a cerebral edema should be given adiffusion enhancing compound such as TSC, e.g., by IV injection orinfusion, or orally, at a dosage in the range of 0.1-2 mg/kg.

In another embodiment, cerebral edema treatment can include one or moreof mannitol, diuretics and corticosteroids. An advantageouscorticosteroid is dexamethasone.

Myocardial Infarction

Myocardial infarction (MI or AMI for acute myocardial infarction),commonly known as a heart attack, occurs when the blood supply to partof the heart is interrupted causing some heart cells to die. This ismost commonly due to occlusion (blockage) of a coronary artery followingthe rupture of a vulnerable atherosclerotic plaque, which is an unstablecollection of lipids (like cholesterol) and white blood cells(especially macrophages) in the wall of an artery. The resultingischemia (restriction in blood supply) and oxygen shortage, if leftuntreated for a sufficient period of time, can cause damage and/or death(infarction) of heart muscle tissue (myocardium).

A diffusion enhancing compound such as a BTCS compounds (e.g. TSC), canbe used, either alone or in conjunction with a thrombolytic, as atreatment for myocardial infarction. A diffusion enhancing compound suchas TSC can be administered by various routes. For example, the compoundwhich can be formulated with other compounds, can be administered at theproper dosage as an intravenous injection or infusion, an intramuscularinjection, or in an oral form. The IV injection route is an advantageousroute for giving a diffusion enhancing compound such as TSC formyocardial infarction since the patient may well be unconscious.Typically, the compound is administered as soon as possible. A patientshowing signs of a myocardial infarction should be given a diffusionenhancing compound such as TSC, e.g., by IV injection or infusion, ororally, at a dosage in the range of 0.1-2 mg/kg.

If a thrombus is believed to be present, a therapeutically effectiveamount, i.e. a clot dissolving amount, of the thrombolytic agent such astPA, can also be administered. Formulations of thrombolytics are wellknown to those skilled in the art. A thrombolytic such as tPA, istypically administered via IV injection. If a diffusion enhancing drughas been administered, the advantage of administration of a thrombolyticis highest within the first ninety minutes, but can extend up to 9 oreven 12 hours after the start of symptoms.

Thrombolytic drugs can be given in combination with intravenous heparin,or low molecular weight heparin, which are anticoagulant drugs.

Deep Vein Thrombosis

Deep vein thrombosis (also known as deep-vein thrombosis or deep venousthrombosis) is the formation of a blood clot (“thrombus”) in a deepvein. It is a form of thrombophlebitis (inflammation of a vein with clotformation).

Deep vein thrombosis commonly affects the leg veins (such as the femoralvein or the popliteal vein) or the deep veins of the pelvis.Occasionally the veins of the arm are affected (if spontaneous, this isknown as Paget-Schrötter disease).

A diffusion enhancing compound such as a BTCS compounds (e.g. TSC), canbe used in conjunction with a thrombolytic as a treatment for deep veinthrombosis. A diffusion enhancing compound such as a BTCS compounds(e.g. TSC), can be used in conjunction with a thrombolytic as atreatment for deep vein thrombosis. A diffusion enhancing compound suchas TSC can be administered by various routes. For example, the compoundwhich can be formulated with other compounds (excipients), can beadministered at the proper dosage as an intravenous injection orinfusion, an intramuscular injection, or in an oral form.

The IV injection route is an advantageous route for giving a diffusionenhancing compound such as TSC for deep vein thrombosis since thepatient may well be unconscious. Typically, the compound is administeredas soon as possible. A patient showing signs of a deep vein thrombosisshould be given a diffusion enhancing compound such as TSC, e.g., by IVinjection or infusion, or orally, at a dosage in the range of 0.1-2mg/kg.

A therapeutically effective amount, i.e. a clot dissolving amount, ofthe thrombolytic agent such as tPA, can also be administered.Formulation of thrombolytics are well known to those skilled in the art.A thrombolytic such as tPA, is typically administered via IV injection.If a diffusion enhancing drug has been administered, the advantage ofadministration of a thrombolytic is highest within the first ninetyminutes, but can extend up to 9 or even 12 hours after the start ofsymptoms.

Thrombolytic drugs can be given in combination with intravenous heparin,or low molecular weight heparin, which are anticoagulant drugs.

Pulmonary Embolism

Pulmonary embolism (PE) is a blockage of the pulmonary artery or one ofits branches, usually occurring when a deep vein thrombus (blood clotfrom a vein) becomes dislodged from its site of formation and travels,or embolizes, to the arterial blood supply of one of the lungs. Thisprocess is termed thromboembolism.

A diffusion enhancing compound such as a BTCS compounds (e.g. TSC), canbe used, either alone or in conjunction with a thrombolytic, as atreatment for pulmonary embolism. The compound can be administered byvarious routes. For example, the compound which can be formulated withother compounds, can be administered at the proper dosage as anintravenous injection or infusion, an intramuscular injection, or in anoral form.

The IV injection route is an advantageous route for giving a diffusionenhancing compound such as TSC for pulmonary embolism since the patientmay well be unconscious. Typically, the compound is administered as soonas possible. A patient showing signs of a pulmonary embolism should begiven a diffusion enhancing compound such as TSC, e.g., by IV injectionor infusion, or orally, at a dosage in the range of 0.1-2 mg/kg.

If a thrombus is believed to be present, a therapeutically effectiveamount, i.e. a clot dissolving amount, of the thrombolytic agent such astPA, is administered. Formulation of thrombolytics are well known tothose skilled in the art. A thrombolytic such as tPA, is typicallyadministered via IV injection. If a diffusion enhancing drug has beenadministered, the advantage of administration of a thrombolytic ishighest within the first ninety minutes, but can extend up to 9 or even12 hours after the start of symptoms.

Thrombolytic drugs can be given in combination with intravenous heparin,or low molecular weight heparin, which are anticoagulant drugs.

The following Examples are illustrative, but not limiting of thecompositions and methods of the present invention. Other suitablemodifications and adaptations of a variety of conditions and parametersnormally encountered which are obvious to those skilled in the art arewithin the spirit and scope of this invention.

EXAMPLES Example 1 Trans Sodium Crocetinate with Tissue PlasminogenActivator in Ischemic Stroke General Description of Animal Model:

Methods: This model uses rabbits and is known as RSCEM. Male New Zealandwhite rabbits were anesthetized using isoflurane (5% induction, 2%maintenance by facemask), the bifurcation of the right carotid arterywas exposed and the external carotid was ligated just distal to thebifurcation, where a catheter was inserted into the common carotid andsecured with ligatures. The incision was closed around the catheter withthe distal ends left accessible outside the neck; the catheter wasfilled with heparinized saline and plugged with an injection cap.Rabbits were allowed to recover from anesthesia for a minimum of 2 huntil they behaved normally. After that time, microclots were preparedfrom blood drawn from a donor rabbit and allowed to clot at 37° C., asdescribed in detail previously [4,10,11]. Microclots were re-suspendedin PBS, then washed and allowed to settle, followed by aspiration of thesupernatant and spiking of the microclots with tracer quantities of15-μm radiolabeled microspheres. The specific activity of the particleswas determined by removing an aliquot, after which appropriate volumesof PBS solution were added so that a predetermined weight of clotparticles were rapidly injected through the catheter and both thesyringe and catheter were flushed with 5 ml of normal saline.

Quantal Dose-Response Analysis: For behavioral analysis, a quantaldose-response data analysis technique was used as described previously[4, 10, 11]. A wide range of lesion volumes is induced to generate bothnormal and abnormal animals with various behavioral deficits. Using 3 ormore different doses of microclots generated each quantal analysiscurve. In the absence of treatment, the low end of the curve (smallnumbers of microclots cause no grossly apparent neurologic dysfunction)and the high end (large numbers of microclots invariably causeencephalopathy or death). Each animal is rated as either normal orabnormal (including dead animals), and inter-rater variability is verylow (<5%). Behaviorally normal rabbits did not have any signs ofimpairment, whereas behaviorally abnormal rabbits had loss of balance,head leans, circling, and/or limb paralysis. With this simple ratingsystem, the composite result for a group of animals is quitereproducible. Briefly, to evaluate the quantitative relationship betweennumbers of clots in the brain and neurological deficits (coma or death),logistic (S-shaped) curves are fitted by computer to the quantaldose-response data (see FIGS. 1 and 2).

These parameters are measures of the amount of microclots (in mg) thatproduce neurologic dysfunction in 50% of a group of animals (P₅₀). TheP₅₀ values are then calculated as described previously [4, 10, 11] andare presented as Mean±SEM. A separate curve is generated for eachtreatment condition tested. A statistically significant shift to theright of the quantal curve or an increase in the P₅₀ value is indicativeof a behavioral improvement and neuroprotection. The data were analyzedusing the t-test.

Specific Study Done:

Drug Treatment: For test substance administration, rabbits were placedin a Plexiglas restrainer for the duration of the treatment. Rabbitswere given a bolus intravenous injection of vehicle or TSC (0.25 mg/kg)over 1 minute using the marginal ear vein at a dose of 0.22 ml/kg. Forthrombolytic studies, tPA (3.3 mg/kg) was given 1 or 3 hourspost-embolization, with 20% as a bolus IV injection over one minute,followed by the remainder infused over 30 min. Genentech, Inc. (SouthSan Francisco, Calif.; Lots 745047, 705409) as described previously [4].For the construction of quantal analysis, rabbits were included in thestudy if they were able to survive to receive treatment followingembolization. All others were excluded from the study and furtheranalysis.

Results: (1) TSC Improves Behavior at 1 Hour Post-Embolization

In this series of studies, the effects of administration of TSC (0.25mg/kg) on behavioral function measured 24 hours following embolizationwas determined. Using the RSCEM, TSC significantly (p<0.05) increasedbehavioral performance with a P₅₀ value of 2.84±1.01 mg (n=24) comparedto the vehicle control (P₅₀=1.01±0.23 mg, n=34), FIG. 1 provides thequantal curve for the effect of TSC on behavior.

(2) TSC Combination Studies: tPA 1 Hour Delay

FIG. 1 also provides a graphical representation of the effects of tPA onbehavior and the group P₅₀ value when given 1 hour followingembolization. tPA significantly improved behavior and increased thegroup P₅₀ value of 2.48±0.17 mg (n=21, p<0.05) compared to the vehiclecontrol group, which had a P₅₀ value of 1.01±0.23 mg (n=34). Incombination studies, when both TSC and tPA were administered 1 hourfollowing embolization, the group P₅₀ value measured 24 hour followingembolization was 3.95±0.73 mg (n=26), a P₅₀ that was significantlydifferent from control (p<0.05). There was a trend for a synergisticeffect of the drug combination that did not reach statisticalsignificance compared to TSC (p=0.372) or tPA (p=0.087).

(3) TSC Combination Studies: tPA 3 Hour Delay

FIG. 2 below provides a graphical representation of the effects of tPAon behavior and the group P₅₀ value when given 3 hours followingembolization. tPA did not significantly improve behavior (p>0.05)resulting in a group P₅₀ value of 1.00±0.56 mg (n=27) compared to thevehicle control group, which had a P₅₀ value of 1.01±0.23 mg (n=34). Incombination studies, when TSC was administered 1 hour followingembolization and tPA was given 3 hours following embolization, the groupP₅₀ value was 2.43±0.24 mg (n=20), a P₅₀ that was significantlydifferent from control (p<0.05).

Summary of Results

FIGS. 1 and 2 show abnormal rabbits as a function of clot weightmeasured in brain. Results are shown as mean±SEM for the number ofrabbits in each group (n). The curve labeled Vehicle (dotted line inFIGS. 1 and 2) shows that 50% of rabbits with a clot dose (P₅₀ value) of1.01±0.23 mg (n=34) are abnormal. TSC (0.25 mg/kg) treatment (solidlines in FIGS. 1 and 2) 1 hour post-embolization increased the P₅₀ valueto 2.84±0.51 mg (n=24, p>0.05). tPA (dashed lines in FIGS. 1 and 2) whenadministered 1 hour post-embolization significantly increased the P₅₀value, but was ineffective when given 3 hours following embolization(FIG. 2, dashed line). The combination of TSC (1 hour) plus tPA (either1 hour, FIG. 1) or (3 hours, FIG. 2) significantly improved behavior andincreased P₅₀ values.

FIG. 3 also shows that TSC administration significantly improvesclinical rating scores when administered within 1 hour of embolization.Moreover, the study shows that TSC may be administered safely incombination with the thrombolytic tPA (FIG. 4) and that combinationtherapy produces a significant behavioral improvement in embolizedrabbits. The data teach that TSC can extend the treatment window for tPAto at least 3 hours in the RSCEM animal model, a time at which tPA aloneis ineffective in this animal model. In addition, the data show that TSCadministered alone at 1 hour post-embolization in this model is alsostatistically effective. Again, it is thought that the times in thisrabbit model can by multiplied by a factor of 3 to 4 in order toestimate human times.

As mentioned previously, tPA is a treatment for heart attacks as well asstrokes. Even though this animal model concerns an ischemic stroke, itmay also offer some suggestions concerning a better treatment for heartattacks caused by clots. For example, the 1-hour data (FIG. 3) show anequal effect of TSC or tPA. In order to determine if those two drugsgiven together would exhibit an even better result, an additional studywas done in which both drugs were injected at a time of 1 hour after theclots were injected into the rabbit brains. Those results are shown inFIG. 5.

All three drug treatments (tPA alone, TSC alone, tPA/TSC combination)provide a statistically-significant result in improving the condition ofthe rabbits. It is also interesting to see that the combination of the 2drugs produced a benefit which appears to be better from either of thedrugs given alone, although it is not statistically different from theindividual drugs given alone. It should be noted, though, that tPA mustbe given within 3 hours of the first symptoms of a stroke, a time whichlimits its use. However, TSC can be given without knowing what type ofstroke has occurred.

Example 2 Trans Sodium Crocetinate Treatment in Hemorrhagic StrokeBackground

A key risk of early intervention in ischemic stroke is that injuryresulting from hemorrhagic stroke and/or hemorrhagic transformationmight be aggravated, as is the case with tissue plasminogen activator(tPA) therapy. Therefore, the current study examined the effect of TSCin a model of intracranial hemorrhage (ICH) in order to evaluate whetherearly TSC treatment under conditions of hemorrhagic stroke adverselyaffects outcomes.

Twelve male, Sprague-Dawley rats (Taconic, Inc.), weighing between 250to 300 Grams were fed ad libitum and maintained on a 12-hour light/darkcycle. The rats were randomly assigned to 1 of 2 groups as follows:TSC-treated group (n=6) receiving intravenous infusion of TSC (totaldose 0.091 mg/kg) and a Control group receiving intravenous infusion of0.9% normal saline.

A collagenase-injection model of ICH was utilized. Animals wereanesthetized for 5 minutes with 5% halothane and endotracheallyintubated. Anesthesia was maintained under 1 to 1.5% halothane withventilation supported using FiO2 of 50%. Tail artery cannulation wasapplied to monitor the blood pressure and arterial blood gas, and arectal temperature probe monitored body temperature. The right femoralvein was cannulated and connected to a microinjection infusion pump forTSC or saline administration. Body temperature was maintained at 37° C.with a heating pad. Rats were secured into a stereotactic frame with amidline incision over the skull and a cranial burr hole of 1 mm indiameter was drilled with 0.2 mm anterior and 3.5 mm right lateral toBregma. A 26-gauge needle was inserted stereotactically into the rightbasal ganglion (5.5 mm ventral) and 5 μL collagenase (0.05 U bacteriacollagenase; type IV, Sigma Chemical Co.) was infused at a rate of 1μL/min using a microinfusion pump. The needle was kept in the striatumfor an additional 5 minutes to limit collagenase flow back into the burrhole. After withdrawing the needle, the craniotomy was sealed with bonewax and the wound was sutured closed.

TSC was injected intravenously 3 hours after ICH at a final total dosageof 0.091 mg/kg. The TSC formulation used was the sterile lyophilized TSCinjectable formulation which was reconstituted with sterile water forinjection and diluted with deionized water pH′d to 8.0 with dilutesodium carbonate. Normal saline (0.9%) given in the equivalent volume asthe TSC dose volume was used as the control vehicle. On randomassignment, either TSC or saline was intravenously infused in theanimals beginning at 3 hours after collagenase injection. Theintravenous dose of TSC administered as an initial bolus injection of0.1 mL followed by an infusion at a rate of 0.01 mL/min for 60 minutes,and a second bolus injection of 0.1 mL given 30 minutes after thecessation of the infusion. After surgery and dosing, the rats wereallowed to recover. Saline-treated animals were used as controls andreceived the same ICH procedure and saline injections (instead of TSC).The outcomes assessed 48 hours post-ICH included hematoma volume,hemoglobin content representing hemorrhagic volume in the injectedstriatum, and tissue edema.

Animals were euthanized 48 hours after collagenase infusion bydecapitation under deep halothane anesthesia. Brains were dissected andsectioned coronally (2 mm thickness). Using Meta Morph image analysissoftware, the hemorrhage area for each section was measured and thetotal hematoma volume was calculated by summing the clot area in eachsection and multiplying by the thickness of the sections. A tissue edemaindex was calculated in each rat by measuring and comparing both of thehemispheres (Edema Index=(R−L)×100/L, R is right hemisphere volume, L isleft hemisphere volume). A hemorrhagic volume index was quantified bymeasuring hemoglobin content in the injected hemisphere. Distilled water(1 mL) was added to the ipsilateral cerebral hemisphere collected fromeach animal, followed by homogenization for 1 minute, sonication on icewith a pulse ultrasonicator for 2 minutes, and centrifugation at 13,000rpm for 30 minutes. After the hemoglobin-containing supernatant wascollected, 800 μL of Drabkin's reagent was added to a 200 μL aliquot andallowed to stand for 15 minutes in the dark. This reaction convertshemoglobin to cyanomethemoglobin, the concentration of which can beassessed by the optical density (OD) of the solution at 550 nmwavelength. Incremental aliquots of blood were obtained from thesaline-treated control group by cardiac puncture after anesthesia. Thisblood was added to freshly homogenized brain tissue obtained fromuntreated rats to generate a standard absorbance curve.

Results

Hematoma Volume/Size after ICH

The influence of TSC on hematoma volume was assessed in acollagenase-injection model of ICH in male rats. FIG. 6 shows examplesof hematomas produced in saline-treated control and TSC-treated rats.

Further assessments demonstrated that TSC does not significantly affecthematoma volume or size after ICH. Hematoma volume was compared betweengroups of saline-treated (n=6) and TSC-treated (n=6) animals. Thehematoma size in the TSC-treated group was slightly, but notstatistically significantly, reduced when compared to the control groupas shown in FIG. 7. Statistical comparisons between groups showed ap>0.05 which was not statistically significant (n.s.) using theStudent's t-test.

Effect of TSC on Hemorrhagic Volume after ICH

TSC-treated animals showed a reduction in hemorrhagic volume after ICHwas assessed. Tissue hemoglobin levels were compared between groups ofsaline-treated control (n=6) and TSC-treated (n=6) animals. Hemorrhagicvolume was reduced by approximately 20% in the TSC-treated group.Statistical comparison showed a significant difference (*statisticallysignificant p<0.05) between the 2 groups using the Student's t-test asnoted in FIG. 8.

Effect of TSC on Tissue Edema after ICH

The influence of TSC on tissue edema was assessed in the ICH model inrats. Tissue edema was compared between groups of saline-treated control(n=6) and TSC-treated (n=6) animals. Tissue edema was reduced byapproximately 45% in the TSC-treated group. Statistical comparisonshowed a significant difference (*statistically significant p<0.05)between groups using the Student's t-test as presented in FIG. 9.

In this model of hemorrhagic stroke, hematoma volume or size did notdiffer between TSC- and saline-treated groups demonstrating that TSCdoes not significantly affect hematoma size after ICH. Hemorrhagicvolume as measured in hemoglobin content was reduced by approximately20% with TSC treatment and this reduction was statistically significant(p<0.05). With TSC treatment, tissue edema was reduced substantially by45% and was statistically significant (p<0.05). These findings areconsistent with the concept that TSC does not aggravate neural injuryafter ICH, and appears to be beneficial.

Thus, TSC may be given to a stroke victim without first ascertaining ifthe stroke is an ischemic one or a hermorrhagic one since it producesbeneficial effects in both kinds of strokes in these animal models.

REFERENCES

-   1. Ingall, T., Stroke-incidence, mortality, morbidity and risk. J    Insur Med, 2004. 36(2): p. 143-52.-   2. Lapchak, P. A. and D. M. Araujo, Advances in ischemic stroke    treatment: neuroprotective and combination therapies. Expert Opin    Emerg Drugs, 2007. 12(1): p. 97-112.-   3. Group, N.r.-P.S.S., Tissue plasminogen activator for acute    ischemic stroke. The National Institute of Neurological Disorders    and Stroke rt-PA Stroke Study Group. N Engl J Med, 1995. 333(24): p.    1581-7.-   4. Lapchak, P. A., D. M. Araujo, and J. A. Zivin, Comparison of    Tenecteplase with Alteplase on clinical rating scores following    small clot embolic strokes in rabbits. Exp Neurol, 2004. 185(1): p.    154-159.-   5. Lapchak, P. A., et al., Neuroprotective effects of the spin trap    agent disodium-[(tert-butylimino)methyl]benzene-1,3-disulfonate    N-oxide (generic NXY-059) in a rabbit small clot embolic stroke    model: combination studies with the thrombolytic tissue plasminogen    activator. Stroke, 2002. 33(5): p. 1411-5.-   6. Lapchak, P. A., et al., Transcranial near-infrared light therapy    improves motor function following embolic strokes in rabbits: An    extended therapeutic window study using continuous and pulse    frequency delivery modes. Neuroscience, 2007. 148(4): p. 907-914.-   7. Lapchak, P. A., J. Wei, and J. A. Zivin, Transcranial infrared    laser therapy improves clinical rating scores after embolic strokes    in rabbits. Stroke, 2004. 35(8): p. 1985-8.-   8. Broderick, J. P., et al., Finding the most powerful measures of    the effectiveness of tissue plasminogen activator in the NINDS tPA    stroke trial. Stroke, 2000. 31(10): p. 2335-41.-   9. Clark, W. M., et al., The rtPA (alteplase) 0- to 6-hour acute    stroke trial, part A (A0276g): results of a double-blind,    placebo-controlled, multicenter study. Thrombolytic therapy in acute    ischemic stroke study investigators. Stroke, 2000. 31(4): p. 811-6.-   10. Lapchak, P. A., Memantine, an uncompetitive low affinity NMDA    open-channel antagonist improves clinical rating scores in a    multiple infarct embolic stroke model in rabbits. Brain Res, 2006.    1088(1): p. 141-7.-   11. Lapchak, P. A., The phenylpropanoid micronutrient chlorogenic    acid improves clinical rating scores in rabbits following multiple    infarct ischemic strokes: synergism with tissue plasminogen    activator. Exp Neurol, 2007. 205(2): p. 407-13.-   12. Lapchak, P. A., et al., Therapeutic window for nonerythropoietic    carbamylated-erythropoietin to improve motor function following    multiple infarct ischemic strokes in New Zealand white rabbits.    Brain Res: 2008. 1238: p 208-14.

It will be readily apparent to those skilled in the art that numerousmodifications and additions can be made to both the present compoundsand compositions, and the related methods without departing from theinvention disclosed.

1. (canceled)
 2. A method of treating a mammal having a myocardialinfarction, pulmonary embolism, or deep vein thrombosis, wherein themethod comprises: i) administering a diffusion enhancing compound tosaid mammal, and ii) administering a thrombolytic agent to said mammal.3-6. (canceled)
 7. A method of treating a mammal having a cerebraledema, wherein the method comprises administering a diffusion enhancingcompound to said mammal.
 8. A method of treating a mammal having a TIA,wherein the method comprises administering a diffusion enhancingcompound to said mammal.
 9. A method as in claim 2, wherein saiddiffusion enhancing compound is a bipolar trans carotenoid.
 10. A methodas in claim 2, wherein said diffusion enhancing compound is a bipolartrans carotenoid salt.
 11. A method as in claim 10, wherein said bipolartrans carotenoid salt is formulated with a cyclodextrin.
 12. A method asin claim 2, wherein said diffusion enhancing compound is TSC.
 13. Amethod as in claim 2, wherein said thrombolytic agent is selected fromthe group consisting of tPA, reteplase, tenecteplase, anistreplase,streptokinase, and urokinase.
 14. A method as in claim 2, wherein saidthrombolytic agent is tPA.
 15. A method as in claim 2, wherein thediffusion enhancing compound is administered within 4 hours of the onsetof symptoms and the thrombolytic agent is administered within 12 hoursof the onset of symptoms.
 16. A method as in claim 2, wherein thediffusion enhancing compound is administered within 3 hours of the onsetof symptoms and the thrombolytic agent is administered within 9 hours ofthe onset of symptoms. 17-18. (canceled)